Terry J. Opgenorth

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

Dissociation Characteristics of Endothelin Receptor Agonists and Antagonists in Cloned Human Type-B Endothelin Receptor

The human type-B endothelin receptor (h-ETB) was cloned from human lung poly A+RNA and stably expressed in CHO cells. Endothelin (ET) receptor binding and stimulation of PI hydrolysis demonstrated that the cloned h-ETB receptor is functional and linked to intracellular signal transduction pathways in CHO cells. The molecular mass of the h-ETB receptor was determined to be 65 KDa, and Bmax and Kd were 0.36 pmol/mg and 80 pM, respectively. Competition studies employing receptor ligands revealed that the potencies of the test ligands (IRL1620, PD142893, and Ro46-2005) were dependent on the length of the incubation time, whereas the natural agonists (ET-1 and ET-3) were not. When competing with ET-1 in the h-ETB receptor binding, the IC50 increased from 1.2 nM to 8.2 nM for IRL1620, 0.068 microM to 1.9 microM for PD142893, and 0.76 microM to 12.7 microM for Ro46-2005, as the incubation time increased from 1 hr to 24 hr. These time-induced changes are likely due to differences in the dissociation characteristics between the artificial ligands and the natural ligands.

Specific Inhibition of Glycosylation by Tunicamycin Affects Endothelin Receptors in Cultured Swiss 3T3 Fibroblasts

The importance of glycosylation to endothelin (ET) receptors was studied in cultured Swiss 3T3 fibroblasts. ET stimulated phosphatidylinositol hydrolysis by 270% in control cells. This stimulatory effect was decreased to < 130% in tunicamycin (TU)-treated cells. TU inhibited protein glycosylation, but did not effect DNA synthesis, cell morphology, or cell numbers. ET-1 binding was greatly reduced in TU-treated cells. In membranes prepared from control cells, [125]ET-1 binding was saturable, and appeared as a straight line in Scatchard analysis with values of 0.7 pmol/mg and 0.9 nM for Bmax and KD, respectively. [125]ET-1 binding to membranes from TU-treated cells was not saturable. Scatchard analysis showed a biphasic line. Values of Bmax, and KD for the higher affinity site were 0.17 pmol/mg and 0.12 nM, respectively. The low affinity site exhibited a much weaker affinity for ET with an estimated KD value of 6.8 nM. Cross-linking studies revealed that the receptor in control membranes was a protein with ...